The effect of some common inactivation procedures on the antigens of bovine herpesvirus 1

Bovine embryonic kidney cells were infected with bovine herpesvirus 1 (BHV1) or were sham-inoculated. When cytopathic effect was apparent, the cells were treated with beta-propiolactone, formalin, heat (56 degrees C), or ultraviolet irradiation until the virus was inactivated. Infected-treated, infected-untreated (IU) and sham-inoculated cultures were solubilized using Triton X-100 detergent. Resulting preparations were tested by 2-dimensional- and fused rocket-immunoelectrophoresis and were evaluated for their ability to inhibit virus neutralization by BHV1 antiserum. Eleven viral antigens were detected consistently in IU preparations, which strongly inhibited virus neutralization. Eight or more IU antigens were detected in beta-propiolactone-treated, formalin-treated and heat-treated preparations; these inhibited virus neutralization less strongly than the IU preparations. No IU antigens were detected in ultraviolet-treated preparations, nor did this material inhibit virus neutralization. One of the IU antigens was reduced preferentially by all treatments. The selective destruction of antigens by the various treatments might allow antigen-specific serological testing to distinguish vaccinated from naturally-exposed cattle.     

Characterization of a factor from bovine intestine that protects against Cryptosporidium parvum infection

Cryptosporidium parvum is a protozoan parasite that causes intestinal infection in a variety of mammals. We have previously described a factor in adult rat or adult bovine intestinal mucosa that protects against C. parvum infection when fed to susceptible infant rats. This factor is absent in intestinal mucosa from bovine calves. In the present study we describe the further characterization of the active component of bovine intestinal mucosa. The ability to protect infant rats against C. parvum infection was found to be associated with the extrinsic membrane protein fraction of the intestinal mucosa. Extrinsic membrane preparations from adult cows, adult rats, and calves were separated by SDS-PAGE. A band with apparent molecular mass of 54 kDa was seen in preparations from adult rat and cow, but not calf. Protein was transferred to PVDF membrane and from this the band was excised and subjected to N-terminal sequence analysis using a gas-phase protein sequenator. A 15-amino acid consensus sequence was generated with homology to leucine aminopeptidase (LAP). Purified LAP was purchased from a commercial source and tested for ability to protect infant rats against C. parvum infection. Rats fed LAP from 7 to 11 days of age and challenged with C. parvum at 9 days were significantly less infected than controls upon necropsy at 15 days of age. These data suggest that a protein with N-terminal sequence homology to LAP may reduce susceptibility of infant rats to C. parvum infection.     

Isolation and characterization of the eighth component of the bovine complement system

OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.     

Bovine interleukin 2: production and characterization

The production of bovine IL 2 was studied and IL 2 was partially characterized. PMA at 5 ng/ml + Concanavalin A at 5 micrograms/ml treatment of peripheral blood mononuclear cells gave a greater yield of IL 2 activity in the supernatants than Con A, PMA or sodium periodate treatments alone. Macrophage depletion increased yields as did the addition of indomethacin, a prostaglandin E2 inhibitor. Bovine IL 2 was sensitive to trypsin, relatively stable at pH 2-9, 2-ME resistant and sensitive to increasing molar concentrations of urea. The activity of bovine IL 2 was reduced by over 45% at 70 degrees C for 30 min and 95% at 90 degrees C for 30 min. Bovine IL 2 was more stable at 4 degrees C than at room temperature and the stability at room temperature could be improved by inclusion of 1% BSA. Bovine IL 2 eluted from DEAE-Sephadex as a broad peak with 0.1-0.2 M NaCl. Peak activity corresponded to a molecular weight of approximately 16,000 daltons on Sephadex G-100.     

Construction of Eukaryotic Expression Vector of GDF-9 Gene and its Effect on the Expression of Expansion-related Genes in Bovine Cumulus Cells

The objective of the study was to construct the eukaryotic expression vector of bovine GDF-9 gene,and investigate its effect on the expression of expansion-related genes in transfected bovine cumulus cells.Eukaryotic expression vector plasmid pcDNA4 myc-his-GDF-9 was constructed by insert GDF-9 in the multiple cloning site.pcDNA4 myc-his-GDF-9 was transfected into bovine cumulus cells by liposome.The expression of GDF-9 gene was detected by Western blotting and the expression of the expansion-related genes in bovine cumulus cells was detected by qRT-PCR.The results showed that GDF-9 could express in transfected bovine cumulus cells and the expression of expansion-related genes PTGS2,PTX3 and HAS2 in transfected bovine cumulus cells were significantly increased (P< 0.05).In a conclusion,the bovine pcDNA4 myc-his-GDF-9 could effectively transfected into bovine cumulus cells,which could help to study farther functions of the GDF-9 gene.     

GDF-9基因真核表达载体的构建及其对牛卵丘细胞扩展相关基因表达的影响

为构建牛生长分化因子9(GDF-9)基因真核表达载体,观察GDF-9基因真核表达载体转染牛卵丘细胞后对卵丘扩展相关基因表达的影响,试验在质粒pcDNA4 myc-his的多克隆位点插入GDF-9基因构建真核表达载体pcDNA4 myc-his-GDF-9,用脂质体转染牛卵丘细胞,利用Western blotting检测了GDF-9蛋白的表达量,同时利用实时荧光定量PCR(qRT-PCR)技术检测牛卵丘细胞中卵丘扩展相关基因的表达量.结果发现,转染了pcDNA4 myc-his-GDF-9的牛卵丘细胞中检测到GDF-9蛋白的表达,且牛卵丘细胞扩展相关基因PTGS2、PTX3和HAS2的表达量显著提高(P< 0.05),表明牛pcDNA4 myc-his-GDF-9可有效地转染到牛卵丘细胞中,该试验结果为进一步研究GDF-9基因的功能奠定了基础.     

Expression of leptin mRNA and CCAAT-enhancer binding proteins in response to insulin deprivation during preadipocyte differentiation in primary cultures of porcine stromal-vascular cells

The aim of this study was to examine the correlation between CCAAT-enhancer binding proteins (C/EBPs) and leptin gene expression in response to insulin deprivation in preadipocytes and adipocytes. Adipose tissue from 7 d-old pigs was digested enzymatically and stromal-vascular (S-V) cells were seeded and plated for 3 d in fetal bovine serum (FBS) with dexamethasone (DEX) followed by 6 d (Days 3–9) in serum-free medium with insulin (850 nM or 10 nM), transferrin, and selenium. During FBS+DEX treatment (Days 0–3) a large number of preadipocytes develop with no lipid accretion. In contrast, preadipocyte number does not change with lipid accretion during insulin treatment (Days 3–9). Total RNA and cells were harvested from S-V cultures after periods with and without insulin after FBS+DEX. Northern-blotting and Western blot analysis were used to study leptin mRNA and C/EBP protein expression in cultures, respectively. Insulin deprivation from Days 3–4 reduced leptin mRNA and C/EBP- protein expression. Treatment with 850 nM or 10 nM insulin from Days 3–9 induced leptin mRNA and C/EBP- expression at a similar level. In cultures treated with 10 nM insulin from Days 3–7, leptin and C/EBP- expression were reduced markedly by insulin deprivation from Days 7–9, but were restored by insulin treatment for 6 hr before harvesting. The restoration of leptin expression by insulin was blocked by cycloheximide treatment. However, C/EBP-β protein levels did not change regardless of insulin deprivation. Insulin deprivation from Days 7–9 in cultures treated with 850 nM insulin from Days 3–7 did not influence C/EBP- or leptin mRNA expression, whereas C/EBP- and leptin expression were reduced after treating these cultures with 1.5 uM okadaic acid for 45 min before harvesting on Day 9. However, cycloheximide treatment for 6 hr before harvesting did not reduce leptin mRNA expression. These results suggest that 1) leptin expression is positively correlated with C/EBP- expression, and 2) the maintenance of leptin expression after insulin deprivation in 850 nM insulin-treated cultures on Day 9 may be associated with the presence of C/EBP- expression and/or activation.     

Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor

A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.     

靶向TLR9基因siRNA的筛选及其对BHV-1增殖规律的影响

为研究牛胚胎气管（bovine embryonic tracheal,EBTr）细胞Toll样受体9（Toll-like receptors 9,TLR9）在牛疱疹病毒Ⅰ型（bovine herpesvirus type 1,BHV-1）感染的天然免疫反应中的作用,本试验利用小分子干扰RNA（siRNA）技术,以TLR9为靶向分别设计并合成3条siRNA干扰序列,用SYBR GreenⅠ实时荧光定量PCR技术检测应用siRNA-TLR9干扰后TLR9基因的表达变化,筛选出最佳的siRNA-TLR9干扰序列,继而转染EBTr细胞使其TLR9基因沉默后感染BHV-1,用TaqMan实时荧光定量PCR方法检测BHV-1不同时间点的增殖变化。结果显示,转染48 h后,与阴性对照组相比,siRNA-TLR9A、siRNA-TLR9B和siRNA-TLR9C分别对TLR9 mRNA表达量下调了60.90%、24.05%和40.75%。筛选出siRNA-TLR9A片段在12~72 h可以显著抑制TLR9 mRNA表达（P < 0.05）。用该片段转染EBTr细胞再感染BHV-1后,6~72 h siRNA-TLR9A组BHV-1 DNA拷贝数低于对照组。本试验成功筛选出了特异性的抑制EBTr细胞TLR9基因的siRNA序列,并证明抑制TLR9的表达可降低BHV-1的增殖能力。     

Interaction of bovine immunoglobulins with complement

Whereas complement (C) in rabbit serum (CR) was bound by bovine antibodies in seven different IgG1 preparations, only two IgG1 preparations could bind the C in guinea pig serum (CGP). Addition of the Clq component of CR to CGP was alone sufficient to render the C-cascade in CGP activable in the presence of bovine erythrocytes sensitized with specific antisera, i.e. reagents. Normal bovine serum was also capable of restoring the haemolytic activity of CGP. However, the bovine serum was much more temperature sensitive than was CR and, as was observed in the sera from MZ twins, it showed considerable variation both in titre values and in prozones when added to CGP.     

Screening of siRNA Targeted for TLR9 Gene and Its Effect on the Proliferation Profile of BHV-1

The aim of this study was to investigate the role of Toll-like receptors 9 (TLR9) of the bovine embryonic tracheal (EBTr) cells in the innate immune response mediated by bovine herpesvirus type 1(BHV-1). Using small interfering RNA (siRNA) technology, three siRNA interference sequences target for TLR9 were designed and synthesized in this study. After siRNA-TLR9 interference, the expression levels of TLR9 gene were detected by Real-time PCR. After 48 h, the expression levels of TLR9 mRNA induced by siRNA-TLR9A, siRNA-TLR9B and siRNA-TLR9C were reduced to 60.90%, 24.05% and 40.75%, respectively. Comparing with the control group, the expression levels of TLR9 mRNA were significantly inhibited by siRNA-TLR9A fragments at 12 to 72 h (P < 0.05), and after transfecting the best fragments and infecting with BHV-1, the BHV-1 DNA copy numbers of siRNA-TLR9A group were lower than BHV-1 DNA of the control group at 6 to 72 h. The specific siRNA fragments target for TLR9 were successfully screened out in this test, and demonstrated that inhibition of TLR9 expressions could reduce the BHV-1 proliferation in EBTr cells.     

Cross-reactions of bovine herpesvirus 1 antigens with those of other cattle herpesviruses

Bovine embryonic kidney cells were infected with bovine herpesviruses (BHV1, 2, or 3), suid herpesvirus 1 (SHV1), or were sham-inoculated. When cytopathic effect was apparent, the cells were solubilized using Triton X-100 detergent. Resulting antigen preparations were tested by 2-dimensional immunoelectrophoresis using bovine fetal serum and antisera directed against BHV1, BHV2, BHV3, SHV1 or a restricted spectrum of BHV1 antigens. Interaction of BHV1 antiserum with BHV1 antigen preparations resulted in 11 precipitation arcs. The same antiserum produced 3 arcs with BHV2, none with BHV3, and 5 with SHV1. The interaction of BHV1 antigen preparations with BHV2, BHV3, or SHV1 antisera failed to produce demonstrable arcs. However, when heterologous antigen or antibody preparations were added to BHV1 homologous 2-dimensional immunoelectrophoresis tests, all 11 BVH1 arcs were modified by BHV1, 2 by BHV2, 4 by BHV3 and 4 by SHV1 preparations. Two antigens were common to the 4 herpesviruses. Antigen preparations were tested for their ability to inhibit virus neutralization by BHV1 antiserum; only the BHV1 preparation was active. Sera were tested for BHV1 neutralizing activity; only BHV1 antiserum and a serum specific for a restricted spectrum of BHV1 antigens were active. A glycoprotein antigen associated with BHV1 neutralization was identified which may be important in the protection of animals against disease.     

Non-immune erythrocyte rosette formation of bovine peripheral blood and thymus lymphocytes

An E-rosetting reaction is described which gave 92.1%±2.4 (mean±S.D.) E-rosettes with bovine fetal thymocytes and 48.2%±8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10% fetal calf serum (FCS). Other media including 100% FCS, MEM with 10% FCS, and RPMI-1640 with 10% FCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 90% (75.2%±9.3) of cells in bovine PBL preparations, and from 90.2 to 97.5% (94.2%±2.1) of cells in bovine fetal thymocyte preparations into T and B cells.     

Isolation and characterization of factor I of the bovine complement system

OBJECTIVE: To isolate and characterize factor I of the bovine complement system. Sample Population-Serum obtained from the blood of beef cattle. PROCEDURES: Serum samples were fractionated to yield factor I by means of sequential precipitation, ion-exchange, and gel-filtration chromatography. The protein was identified throughout the procedure on the basis of its ability to degrade the alpha'-chain of bovine C3b in the presence of bovine factor H. Electrophoresis in polyacrylamide gels was used to assess the degradation of C3b and determine the molecular weights of factor I and its component polypeptide chains. RESULTS: Bovine factor I had an apparent molecular weight of 94 kd and consisted of 2 disulfide-bonded polypeptides that had apparent molecular weights of 51 and 42 kd (under reducing conditions). Factor H was required for the factor I cleavage of the alpha'-chain of bovine C3b into iC3b. A similar cofactor effect was provided by trypsinized bovine erythrocytes or erythrocyte ghosts. Bovine properdin was prepared and shown to be a single polypeptide chain of 58 kd in the reduced form. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine factor I can be purified from serum by a simple 4-step procedure. It is structurally and functionally comparable to factor I of other species, and its purification completes the isolation and characterization of all the soluble components of the bovine alternative complement pathway.     

Effect of macrophages and in vitro infection with parainfluenza type 3 and respiratory syncytial viruses on the mitogenic response of bovine lymphocytes.

Bovine blood lymphocytes, depleted of macrophages by absorption on plasma-gelatin coated plastic flasks, followed by passage through Sephadex G-10 columns, failed to respond to pokeweed mitogen stimulation. Adherent monocytes or alveolar macrophages added to purified lymphocyte preparations at 10% or less were able to restore the transformation response. Exposure of alveolar macrophages or purified lymphocytes to 2 bovine respiratory syncytial virus strains for 24 hours substantially reduced the transformation response when mixed with uninfected lymphocytes or macrophages. Exposure of alveolar macrophages or purified lymphocytes to 2 bovine parainfluenza type 3 virus strains produced a similar reduction in activity after 48 hours. Heat inactivation of the viruses removed their inhibitory ability. Immunofluorescence studies revealed that both alveolar macrophages and lymphocytes were permissive for parainfluenza type 3 virus, whereas only a small number of alveolar macrophages and lymphocytes were infected with respiratory syncytial virus. The results suggest that both viruses are capable of adversely affecting the interaction between macrophages and lymphocytes, although the mechanisms by which this is achieved may be different.     

Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes

Assay conditions were established for the fatty acid elongation and the delta 9 desaturase enzyme systems of bovine liver and adipose tissue microsomes; rat liver microsomes were used as a reference. Overall fatty acid elongation was determined by measuring the incorporation of [2-14C]malonyl-coenzyme A (CoA) to 14C-labeled stearate. Rat liver elongation activity was .50 +/- .02 nmol.min-1.mg protein-1; bovine liver microsomal elongation activity was substantially lower (P less than .05), with a mean value of .15 +/- .02 nmol.min-1.mg protein-1. The elongation activity of bovine s.c. adipose tissue microsomes (.42 +/- .10 nmol.min-1.mg protein-1) was not different (P greater than .05) from the activity observed in rat liver microsomes. To determine the fatty acid delta 9 desaturase activity, microsomes were incubated in the presence of [1-14C]stearoyl-CoA and nicotinamide adenine dinucleotide (reduced form) (NADH), and the production of radioactively labeled oleate was quantified. Microsomal delta 9 desaturase activity was similar in rat liver and bovine s.c. adipose tissue microsomes with rates of .15 +/- .04 and .21 +/- .05 nmol.min-1.mg protein-1, respectively. However, no desaturase activity was detected in bovine liver microsomes, indicating that the liver is not a major site of oleate synthesis in this species. To investigate differences in fatty acid metabolism relative to breed type, eight Angus and seven Braford heifers were slaughtered at approximately 12 mo of age. Subcutaneous fat thickness over the 12th-13th thoracic vertebrae was greater in the Angus heifers than in the Braford heifers. However, no differences (P greater than .05) were observed in mean adipocyte size or number of cells per gram of adipose tissue between the Angus and Braford heifers. Similarly, there were no significant differences between the Angus and Braford s.c. adipose tissues for microsomal fatty acid elongation or delta 9 desaturation, or for nicotinamide adenine dinucleotide phosphate (NADP)-malate dehydrogenase, fatty acid synthetase, or the pentose cycle reductases. The inability of bovine liver to convert stearate to oleate was in agreement with the fatty acid composition of the liver lipid, which had a smaller percentage of oleate and a higher percentage of stearate than s.c. adipose tissue.     

Effect of storage on measurement of ionized calcium and acid-base variables in equine, bovine, ovine, and canine venous blood.

The stability of blood ionized calcium (Ca2+) and acid-base variables in equine, bovine, ovine, and canine venous blood samples (n = 15, in each group) stored at 4 C for 3, 6, 9, 24, or 48 hours was studied. Variables included blood Ca2+ and standard ionized calcium (Ca2+ corrected to pH 7.4) concentrations, pH, blood carbon dioxide and oxygen tensions, base excess, bicarbonate concentration, and total carbon dioxide content. Results indicate that storage of blood samples at 4 C for up to 48 hours, despite appreciable acid-base changes, is associated with less than 1.5% change in equine, bovine, and ovine blood Ca2+ concentrations. Similar changes were observed in canine blood during the first 9 hours' storage. After 24 and 48 hours' storage, clinically relevant decrease (10.5 and 15.5%) in canine blood Ca2+ concentration was measured. Therefore, Ca2+ concentration in equine, bovine, and ovine venous blood samples stored up to 48 hours, and in canine blood samples stored up to 9 hours at 4 C is of diagnostic use.     

Comparison of bovine IgG1, IgG2 and IgM for ability to promote killing of Mycoplasma bovis by bovine alveolar macrophages and neutrophils

The IgG1, IgG2 and IgM fractions were purified by chromatography from bovine antisera to Mycoplasma bovis. They were assayed for specific antibody and compared for ability to promote killing of M. bovis by bovine peripheral-blood neutrophils and alveolar macrophages. None of the Ig preparations killed the mycoplasma in the absence of the cells. The IgG1 and IgG2 preparations both promoted mycoplasma killing by the macrophages; IgM appeared to have no effect. The IgG2 preparation promoted killing by the neutrophils but neither the IgG1 or IgM fraction appeared effective.     

Development and evaluation of an enzyme-linked immunosorbent assay for quantification of the humoral response of cattle vaccinated against Campylobacter fetus

OBJECTIVE: To develop a reliable ELISA by use of a unique antigen preparation for serum IgG quantification after vaccination against Campylobacter fetus in cattle. ANIMALS: Twenty-six 24-month-old virgin Hereford heifers and a naturally infected Hereford bull. PROCEDURES: 5 antigens were prepared from a cell suspension of C fetus. Antigen preparations were the same as those reported in the literature, with the exception of antigens that were obtained by detergent solubilization of a C fetus cell suspension. For each antigen preparation, the optimal ELISA conditions for its immobilization were determined. Biotinylated antibodies against bovine immunoglobulins were obtained and used in the ELISA. Two groups of heifers were inoculated with commercial vaccines according to manufacturers' instructions. A control group was included. The immune response of vaccinated heifers and controls was followed for 6 months. RESULTS: Detergent solubilized C fetus antigens resulted in better ELISA performance than other antigen preparations. Antigens were optimally immobilized at neutral pH and low ionic strength. All antigen preparations saturated the well with the same amount of protein. The vaccination schedule that advised a booster resulted in higher antibody titers, which were sustained over a longer period than the other schedule. CONCLUSIONS AND CLINICAL RELEVANCE: In the vaccination of cattle against C fetus, the ELISA we have developed may be used to evaluate serum antibody concentrations in response to various vaccines and vaccination schedules. Our results indicate that it is advisable to include a booster in the immunization protocol.     

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.